Tuberculosis has resurged dramatically in recent years in the United States. An estimated 51,700 excess tuberculosis cases have been recorded through 1992. Commensurate with the rise in tuberculosis frequency, an increase in the isolation of Mycobacterium tuberculosis strains resistant to one or more antibiotics commonly used as treatment has occurred. Together, these disease trends represent a clear and present danger to society. The long term goal of the proposed studies is to develop DNA-based diagnostic strategies for rapid identification and drug susceptibility testing of M. tuberculosis in primary clinical samples such as sputum. Specifically, the aims of the research are to, (i) Employ automated DNA sequencing to rapidly and unambiguously define the type and frequency distribution of mutations in several genes strongly associated with resistance to anti-tuberculosis medications, including rifampin, isoniazid, ethionamide, streptomycin, and ciprofloxacin. (ii) Explore the ability of an emerging technology, termed "sequencing by hybridization," or "matrix sequencing," to rapidly diagnose tuberculosis and identify mutations associated with drug resistance in primary clinical specimens and early positive cultures. (iii) Test the feasibility of using automated DNA sequencing strategies to rapidly diagnose tuberculosis and infections caused by mycobacteria other than tuberculosis, by characterization of species-specific allelic polymorphism in the structural gene (hsp65) encoding a heat shock protein, and identify mutations associated with drug resistance in M. tuberculosis. Both primary clinical specimens and early positive cultures will be used in the context of a busy hospital mycobacteriology diagnostic laboratory. Successful accomplishment of the proposed research will demonstrate the capability of diagnosing infection with M. tuberculosis, and other mycobacterial species, and identifying drug resistant M. tuberculosis strains in primary specimens in less than 24-hours.